Principle of HPLC
HPLC is abbreviated from the term High-Performance Liquid Chromatography. It is a well-accepted technique for the separation of multiple substances in a single test. HPLC analysis techniques are used in the determination of the assay of the raw material or finished products. This technique is also suitable for identification tests with comparison to a known reference standard. In the identification process, there are many factors that can be identified like retention time (Tr) Area of Peak and the shape of the peak. Thus, HPLC is the key instrument of modern pharmaceutical laboratories. The principle of HPLC is illustrated with the complete instrumentation of HPLC.
Principle of HPLC
The principle of HPLC may be described as the separation of substances with the help of a mobile phase and a stationary phase that is packed in a column. In this technique, both qualitative and quantitative analysis can be performed.
When the substance is dissolved in a particular mobile phase which is usually water or Acetone, an Alcohol-based solution. The analyte prepared in the mobile phase is injected into the system, then the analyte passes through the column then the analyte is separated and detected by the associated UV-Vis detector, which registers the time and amount of the substance. The detector converts the information into statics units with the help of a computer system. These values are calculated and the assay of substance is found.
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Role of HPLC Column
The column is the key part of the HPLC system. quality and efficiency of the column decide the results of the analysis. The whole separation process is carried out inside the column, when the analyte is passed through the column with the help of high pressure then the compound separation process occurs in the column.
Different parameters of the column decide the retention time, separation, and accuracy of the analysis. The packing of the column ensures the separation of a particular substance. Every molecule can not be separated by any column. The length and diameter of a column impact the retention time of the molecule.
There are different types of columns are used for different columns. Some more significant terms have a role in the chromatographic system like normal-phase, reversed-phase, size exclusion, ion exchange, affinity, and hydrophilic interaction.
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Types of column:
In pharmaceutical laboratories, lots of types of columns are used. This depends on the type of molecule to be tested, the type of column required to use in that testing. There are four main types of columns:
- Normal Phase Columns
- Reverse Phase Columns
- Ion Exchange Columns
- Size Exclusion Columns
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The detector’s function is to register the time and amount of analyte in form of an area. The perceived change by the detector is converted into electronic signals that are computed by an associated PC. Usually, UV-VIS detectors are used in the pharmaceutical industry HPLCs.
Gradient or Isocratic HPLC system:
There are two types of HPLC that are used in the pharmaceutical industry that is Isocratic or Gradient or Binary system. The main difference between these two is the supply of the mobile phase to the system. A constant mobile phase consumer is Isocratic, and in a binary system, more than one mobile phase can be run simultaneously. In a gradient system facility of change in composition is allowed. The pressure of two different mobile phases can be different as per the need of the condition of the analysis process.
The analyte separated by column with help of the mobile phase is recorded by the detector as signal peaks. The total area or the amount of peaks is known as a chromatogram. The qualitative and quantitative data of the analyte are provided by every peak. The area of peak represents the assay or the concentration of an analyte in the mixture. Few more technical terms are important in chromatography.
The time required to bring the analyte compound from injection to detector is called delay time.
The retention time is the time required between the instant of sample injection to the time of detection of the analyte, and the meantime of the peak formation. That means a total time of peak formation. The retention time of any analyte gets imprinted in the peak graph.
The peak width counted between the arrival of the peak from baseline to falling flat to baseline again after the formation of a valid peak.
The tailing factor is the difference in the degree of tailing to the starting of the peak. In other words, the extent of asymmetry of the peak is the tailing factor that should not be more than 2% generally of the total chromatogram of substance. T= b/a, where a is the width of the front half of the peak, and b= width of the back half of the peak. All the values are measured at 10% of the height of the peak.
The pump is an integral part of the HPLC system. The pump is used to flow the mobile phase through the column and system. The pump provides a stable and uniform pressure throughout the whole process. The pressure can be adjusted high or low accordingly to the requirement. In liquid chromatography systems reciprocating pumps are used largely.
The injector is fitted on the pump. The eluent is injected with the conventional method with a syringe. Modern days autosamplers are also used in some industries.
Degasser is used to de-gasify the mobile phase and eluents solution. The mobile phase and analyte solution have some invisible bubbles which may contain air in between them, those small bubbles may affect the results to some extent. To rectify the possibility of error degasser is used. The mobile phase containing air bubbles may produce turbulent in the baseline.
Adavantages of HPLC:
- Speed of testing
- Efficiency of test
- Accuracy of test
Liquid chromatography is a very broad chapter that can not be limited to just the principle of HPLC. The complete instrumentation HPLC has its own working principle and standards. HPLC is the modern-day most trusted analysis method. Increasing day by day a load of audits and to meet the compliances of different validation plans, HPLC is a must-have instrument for every pharmaceutical laboratory. Liquid chromatography is also a requirement of all pharmacopeial standards. Every raw material and dosage form HPLC testing is demanded in the individual monographs. HPLC testing defeats all the traditional chemical methods of analysis due to its multi-dimensional identification capabilities of substances. HPLC is the most sophisticated instrument that requires a higher level of alertness during performing the tests. It is very important to calibrate the HPLC system regularly.